The invention relates to an improved method for preventing or treating vascular hemorrhaging. More specifically, the invention concerns methods for providing improved thrombolytic therapy to individuals who receive such therapy, and for diagnosing and treating diseases, such as Alzheimer""s Disease, that are characterized by the deposition of amyloid deposits. The invention further relates to therapeutic agents for the prevention of the vascular and cellular damage induced by amyloid deposits. This invention was funded with Government funds (R01AG10462 and R01AG11525). The Government has certain rights in this invention.
The serum protein, plasminogen, plays an integral role in the proteolytic dissolution (or fibrinolysis) of blood clots. Plasminogen is an inactive xe2x80x9cproenzyme.xe2x80x9d It has a specific affinity for fibrin, and thus becomes incorporated into blood dots as they form. Plasminogen""s proteolytic activity is released by xe2x80x9cplasminogen activatorsxe2x80x9d (xe2x80x9cPAxe2x80x9d) that specifically cleave the molecule to yield the active protease, plasmin. Plasmin is capable of digesting the fibrin threads of blood clots, as well as other substances involved in creating blood clots, such as fibrinogen, factor V, factor VIII, prothrombin, and factor XI (for review, see Danxc3x8, K. et al., Adv. Canc. Res. 44:139-266 (1985), herein incorporated by reference)).
Plasmin is a serine protease, and exhibits substantial amino add and mechanistic homology with trypsin, chymotrypsin, and pancreatic elastase. Plasmin has a relatively broad trypsin-like specificity, hydrolyzing proteins and peptides at lysyl and arginyl bonds (Castellino, R. W. et al., Meth. Enzymol. 80:365-380 (1981); Danxc3x8, K. et al., Adv. Canc. Res. 44:139-266 (1985)).
Two classes of natural mammalian plasminogen activators have been described: urokinase-type plasminogen activator and tissue-type plasminogen activator (xe2x80x9ct-PAxe2x80x9d) (Danxc3x8, K. et al., Adv. Canc. Res. 44:139-266 (1985); Devlin, et al., PCT appl. WO88/05081; Kasaia et al., U.S. Pat. No. 5,098,840; Hayashi, S. et al., U.S. Pat. No. 4,851,345; Sasaki et al., U.S. Pat. 4,258,030; Hayashi, S. et al., U.S. Pat. No. 5,004,609; Pyke, C. et al., Amer. J. Pathol. 138:1059-1067 (1991); Madison, E. L. et al., Nature 339:721-724 (1989); Blasi, F. et al., J. Cell. Biol. 104:801-804 (1987)). These two classes of molecules can be distinguished immunologically, by tissue localization, and by the stimulation of their activity by fibrin. In addition, a third plasminogen activator, streptokinase, has also been described. Streptokinase differs from urokinase and t-PA in that it is a bacterial protein produced by the streptococci.
Urokinase-type plasminogen activator (UK) is a multi-domain protein with one domain being a trypsin-like serine protease (Castellino, R. W. et al., Meth. Enzymol. 80:365-380 (1981); Danxc3x8, K. et al., Adv. Canc. Res. 44:139-266 (1985); Straxcex2burger, W. et al., FEBS Lett. 157:219-223 (1983)). This protease domain converts plasminogen to plasmin by cleavage at an arginyl residue (Castellino, R. W. et al., Meth. Enzymol. 80:365-380 (1981); Danxc3x8, K. et al., Adv. Canc. Res. 44:139-266 (1985)). The amino add sequence and three-dimensional structure of several serine proteases, including trypsin, chymotrypsin, and elastase have been deduced (Danxc3x8, K. et al., Adv. Canc. Res. 44:139-266 (1985); Straxcex2burger, W. et al., FEBS Lett. 157:219-223 (1983)).
Urokinase is synthesized in the kidneys, and can be recovered from urine. It is initially produced as a single chain protein, xe2x80x9cpro-urokinasexe2x80x9d that can be proteolytically cleaved by plasmin into an active two-chain protein (Devlin, et al., PCT appl. WO88/05081).
Tissue-type plasminogen activator (t-PA) is produced by the cells that line the lumen. of blood vessels or endothelial cells. Like urokinase, t-PA is also initially produced as a single-chain molecule (Rijken, D. G. et al., J. Biol. Chem. 256:7035-7041 (1981); Pennica, D. et al., Nature 301:214-221 (1983)).
The known plasminogen activators differ significantly in characteristics such as their biological half-lives and their preference for fibrin. All three classes of activators have been widely used as thrombolytic agents for the treatment of thrombosis in myocardial infarction, stroke, arterial occlusion, etc. (Kasai et al., U.S. Pat. No. 5,098,840; Hayashi et al., U.S. Pat. No. 5,004,609; Hayashi et al., U.S. Pat. No. 4,851,345; Sasaki et al., U.S. Pat. No. 4,258,030).
The administration of t-PA for the treatment of thrombosis in myocardial infarction, stroke, arterial occlusion, and other cardiovascular diseases reflects the production of minute blood clots which are formed during the disease process. The presence of such clots significantly increases the criticality of the disease, and increases its morbidity. Since t-PA is able to activate plasminogen to plasmin, it is capable of initiating the cascade of events needed to dissolve undesired blood clots. As such, its administration significantly decreases the mortality associated with myocardial infarction and other acute cardiovascular conditions.
Unfortunately, the use of t-PA and streptokinase has been associated with the occurrence of hemorrhages in some individuals (Pendlebury, W. W. et al., Annls. Neurol. 28:210-213 (1989); Wijdicks, E. F. M. et al., Stroke 24:554-557 (1993); Kase, C. S. et al., Annls. Intern. Med. 112:17-21 (1990); Molinari, G. F. Stroke 24:523-526 (1993);), particularly when administered with anti-clotting factors such as coumarin or heparin. This phenomenon has limited the use of t-PA and streptokinase to treat cardiovascular disease in certain classes of patients, notably, the elderly (Topol, E. J. et al., New Engl. J. Med. 327:45-47 (1992); De Jaegere, P. P. et al., J. Amer. Col. Cardiol. 19:289-294(1992); Gore, J. M. et al., Circulation 183:448-459 (1991)).
Alzheimer""s Disease (xe2x80x9cADxe2x80x9d) is a progressive disease of the human central nervous system. It is manifested by dementia in the elderly, by disorientation, loss of memory, difficult with language, calculation, or visual-spatial skills, and by psychiatric manifestations. It is associated with degenerating neurons in several regions of the brain. Alzheimer""s Disease is reviewed by Price, D. L. et al. (Clin. Neuropharm. 14:S9-S14 (1991)); Pollwein, P. et al. (Nucl. Acids Res. 20:63-68 (1992)); Regland, B. et al. (Med. Hypoth. 38:11-19 (1992)) and Johnson, S. A. (In: Review of Biological Research in Aging, Vol. 4., Rothstein, M. (Ed.), Wiley-Liss, NY, 163-170 (1990)).
Pathologically, Alzheimer""s Disease is recognized by the presence of intracellular tangles, and an extracelluar 39-43 amino add peptide known as the xcex2/A4amyloid peptide (Price, D. L. et al., Clin. Neuropharm. 14:S9-S14 (1991); Podlisny, M. B. et al., Science 238:669-671 (1987); Currie, J. R. et al., J. Neurosci. Res. 30:687-689 (1991)). The fibrils formed by this peptide are concentrated in amyloid deposits in the extracellular space of the brain parenchyma and in the vascular elements of the brain and the pia-arachnoid (Currie, J. R. et al., J. Neurosci. Res. 30:687-689 (1991)). All cases of Alzheimer""s Disease show such deposition of amyloid in brain parenchyma.
The amyloid peptide is produced from the proteolytic cleavage of an amyloid precursor protein (xe2x80x9cAPPxe2x80x9d) which is encoded by the APP gene located on chromosome 21. The APP gene is preferentially expressed in the brain cells of the central nervous system. APP mRNA is processed by alternate splicing, and by proteolytic cleavage, such that different isoforms of APP are generated (Pollwein, P. et al. (Nucl. Acids Res. 20:63-68 (1992); Price, D. L. et ad., Clin. Neuropharm. 14:S9-S14 (1991)).
Researchers have proposed that APP is a cell surface receptor or a transmembrane protein, in which the xcex2/A4 domain is partly embedded in the cell membrane. The secretion of the xcex2/A4 peptide thus reflects the cleavage of the domain from the precursor molecule (see, Roch, J. M. et al., J. Biol. Chem. 267:2214-2221 (1992)). Although the accumulation of xcex2/A4 peptide in Alzheimer""s Disease is believed to result from the so-called xe2x80x9camyloidogenicxe2x80x9d processing of one or more of the APP isoforms (Currie, J. R. et al., J. Neurosci. Res. 30:687-689 (1991)), the exact mechanism of xcex2/A4 peptide formation is not yet known (see, Johnson, S. A. (In: Review of Biological Research in Aging, Vol. 4., Rothstein, M. (Ed.), Wiley-liss, NY, 163-170 (1990); Roch, J. M. et al., J. Biol. Chem. 267:2214-2221 (1992)).
The deposition of fibrils of xcex2-amyloid peptide in the brain, in the form of neuritic deposits or within the walls of blood vessels, is a characteristic feature of a number of disorders including Alzheimer""s Disease, Hereditary Cerebral Hemorrhage With Amyloidosis-Dutch type (xe2x80x9cHCHWA-Dxe2x80x9d), Down""s syndrome and cerebral amyloid angiopathy (xe2x80x9cCAAxe2x80x9d). xcex2-amyloid deposition also occurs in normal aging (Glenner, G. G. et al., Biochem. Biophys. Res. Commun. 120:885-890 (1984); Masters, C. L. et al., Proc. Natl. Acad. Sci. (U.S.A.) 82:4245-4249 (1985); van Duinen, S. G. et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5991-5994 (1987); Coria, F. et al., Amer. J. Pathol. 129:422-428 (1987); Prelli, F. et al., J. Neurochem. 51:648-651 (1988)). In CAA and especially in HCHWA-D, where the amyloid deposits are predominantly in the blood vessels, brain hemorrhage is a frequent event (Vonsattel, J. P. G. et al., Annls. Neurol. 30:637-649 (1991)).
To date there is no treatment for Alzheimer""s Disease at any stage of its development. Two therapeutic reagents, Cognex and Menthane, appear to give slight relief to some victims but do not alter the course of the disease.
In view of the importance of diagnosing, predicting, and treating Alzheimer""s Disease, an effective means for achieving these goals would be highly desirable. It would further be desirable to provide an improved thrombolytic therapy that would prevent or lessen the risk of undesired hemorrhage. The present invention provides reagents and methods for accomplishing such improved diagnosis and therapy.